Characterization of 2-[125I]iodomelatonin binding sites in Syrian hamster peripheral organs.
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The neurohormone melatonin is a key agent in synchronizing the circadian rhythms. At least three types of binding sites have been described for melatonin: the G-coupled, seven-transmembrane domain receptors mt1 and MT2 and a putative binding site called MT3. The latter has been described in hamster brain membranes, and its binding capacity is optimum at 4 degrees C. We further characterized this binding site on other peripheral hamster tissues, including intestine, liver, kidney, lung, muscle, and heart. We found a high level of binding sites (>30 fmol/mg of protein) in intestine and kidney. Furthermore, we completed the existing pharmacological profile of this site, which can now be described as 2-iodomelatonin > 6-chloromelatonin > methy-isobutyl-amiloride > acridine orange > 5-methylcarbonylamino-N-acetyltryptamine > prazosin > N-acetylserotonin > melatonin. This profile was found in all the hamster organs tested that had a large number of binding sites, namely, brain, intestine, kidney and liver. Furthermore, when comparisons were possible, the MT3 pharmacological characteristics were similar to those described in the literature for hamster brain and testis. This profile was compared to the pharmacology obtained on human cloned mt1 and MT2 receptors and proved to be completely different, as expected. We provide new evidence for an alternate melatonin binding site not only in hamster brain but also in some peripheral organs. (+info)
Pentoxifylline-stimulated capacitation and acrosome reaction in hamster spermatozoa: involvement of intracellular signalling molecules.
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We investigated the role of cAMP/cGMP, protein kinases and intracellular calcium ( [Ca2+]i) in pentoxifylline-stimulated hamster sperm capacitation and the acrosome reaction (AR) in vitro. Treatment with pentoxifylline (0.45 mM) initially increased sperm cAMP values 2.8-fold, compared with untreated controls (396 +/- 9.2 versus 141 +/- 6.0 fmoles/10(6) spermatozoa; mean +/- SEM, n = 6) after 15 min, although by 3 h, cAMP values were similar (503-531 fmoles/10(6) spermatozoa). cGMP values ( approximately 27 fmoles/10(6) spermatozoa) were the same in treated and control spermatozoa. Both sperm capacitation and the AR, determined from the absence of an acrosomal cap, were stimulated by pentoxifylline; these were almost completely inhibited by a Cl-/ HCO3- antiporter inhibitor (4,4-diisothiocyanato-stilbene-2,2 disulphonic acid; 1 mM) defined from the degree of sperm motility and by a protein kinase A inhibitor (H89; 10 microM). A protein kinase C inhibitor (staurosporine, 1 nM) did not affect pentoxifylline-stimulated capacitation but inhibited the AR by 50%. A protein tyrosine kinase inhibitor (tyrphostin A-47, 0.1 mM) had no effect on either pentoxifylline-stimulated capacitation or AR. A phospholipase A2 inhibitor (aristolochic acid, 0.4 mM) markedly inhibited the pentoxifylline-stimulated AR but not capacitation. When intracellular sperm calcium [Ca2+/-]i was measured using fura-2-AM, there was an early rise (271 nM at 0.5 h) in pentoxifylline-treated spermatozoa; this appeared to be due to intracellular mobilization rather than to uptake. In the absence of extracellular Ca2+, sperm motility was maintained in the presence of pentoxifylline, but capacitation did not occur; spermatozoa exhibited a low level of hyperactivated motility and had a poor rate of AR (20.5 +/- 2.3%). These results suggest that: (i) the pentoxifylline-stimulated early onset of sperm capacitation may be mediated by an early rise in cAMP and [Ca2+/-]i and involves protein kinase A activity; and (ii) pentoxifylline-stimulated AR may require phospholipase A2 and protein kinase C activity. (+info)
Regulation of glycosphingolipid metabolism in liver during the acute phase response.
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The host response to infection is associated with multiple alterations in lipid and lipoprotein metabolism. We have shown recently that endotoxin (lipopolysaccharide (LPS)) and cytokines enhance hepatic sphingolipid synthesis, increase the activity and mRNA levels of serine palmitoyltransferase, the first committed step in sphingolipid synthesis, and increase the content of sphingomyelin, ceramide, and glucosylceramide (GlcCer) in circulating lipoproteins in Syrian hamsters. Since the LPS-induced increase in GlcCer content of lipoproteins was far greater than that of ceramide or sphingomyelin, we have now examined the effect of LPS and cytokines on glycosphingolipid metabolism. LPS markedly increased the mRNA level of hepatic GlcCer synthase, the enzyme that catalyzes the first glycosylation step of glycosphingolipid synthesis. The LPS-induced increase in GlcCer synthase mRNA levels was seen within 2 h, sustained for 8 h, and declined to base line by 24 h. LPS-induced increase in GlcCer synthase mRNA was partly accounted for by an increase in its transcription rate. LPS produced a 3-4-fold increase in hepatic GlcCer synthase activity and significantly increased the content of GlcCer (the immediate product of GlcCer synthase reaction) as well as ceramide trihexoside and ganglioside GM3 (products distal to the GlcCer synthase step) in the liver. Moreover, both tumor necrosis factor-alpha and interleukin-1beta, cytokines that mediate many of the metabolic effects of LPS, increased hepatic GlcCer synthase mRNA levels in vivo as well as in HepG2 cells in vitro, suggesting that these cytokines can directly stimulate glycosphingolipid metabolism. These results indicate that LPS and cytokines up-regulate glycosphingolipid metabolism in vivo and in vitro. An increase in GlcCer synthase mRNA levels and activity leads to the increase in hepatic GlcCer content and may account for the increased GlcCer content in circulating lipoproteins during the acute phase response. (+info)
Hamsters and guinea pigs differ in their plasma lipoprotein cholesterol distribution when fed diets varying in animal protein, soluble fiber, or cholesterol content.
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There were two objectives to these studies: 1) to compare the lipoprotein cholesterol distribution in two animal models in response to different dietary treatments and 2) to assess whether the hypercholesterolemia induced by high cholesterol intake could be reversed by consumption of vegetable-protein and/or dietary fiber. Guinea pigs, which carry the majority of plasma cholesterol in LDL, and hamsters, with a higher distribution of cholesterol in HDL, were evaluated in three different studies. In Study 1, animals were fed semi-purified diets for 4 wk with proportions of 60:40, 20:80 or 0:100 (w/w) of casein/ soybean protein. Hamsters and guinea pigs that consumed 100% soybean protein had lower plasma total cholesterol (TC) than those fed diets containing casein (P < 0.01). In Study 2, three doses of dietary pectin (2.7, 5.4, or 10.7 g/100g) added in place of cellulose were tested. Intake of 10.7 g/100 g pectin resulted in the lowest plasma TC concentrations for both species (P < 0.01). Although the TC lowering was similar in studies 1 and 2, the lipoprotein cholesterol distribution differed. Whereas the differences in plasma cholesterol were in LDL in guinea pigs, hamsters exhibited differences in both non-HDL and HDL cholesterol. In study 3, animals were fed 100% soybean protein, 10.7 g/100 g pectin, and three doses of dietary cholesterol: 0.04, 0.08, or 0.16 g/100 g, which is equivalent to 300, 600, or 1,200 mg/d in humans. Guinea pigs and hamsters had the highest plasma LDL and hepatic cholesterol concentrations when they consumed 0.16 g/100 g of cholesterol (P < 0.01). However, intake of 0.08 g/100 g of cholesterol resulted in lower plasma LDL cholesterol concentrations than did consuming high animal protein (60:40 casein/ soy) or low soluble fiber (2.7 g/100 g). Relatively high levels of dietary cholesterol combined with vegetable protein and soluble fiber resulted in desirable lipoprotein profiles in animal models that significantly differ in their lipoprotein cholesterol distribution. (+info)
Late increase of serum S100 beta protein levels in hamsters after oral or intraperitoneal infection with scrapie.
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Following recent reports of elevated serum S100 beta protein (S100 beta) levels in patients with genetic and sporadic Creutzfeldt-Jakob disease and in rodents parenterally infected with scrapie, the suitability of serum S100 beta as a preclinical marker for transmissible spongiform encephalopathies was assessed in time-course studies. Syrian hamsters were orally and intraperitoneally challenged with scrapie and assayed for serum S100 beta levels at various times after infection. Although elevated serum S100 beta levels were consistently observed in terminally ill animals for both routes of infection, the experiments failed to detect significantly increased S100 beta serum concentrations prior to the manifestation of clinical symptoms. Thus, in this animal model, serum S100 beta does not appear to be an appropriate marker for the preclinical detection of scrapie, but it may provide a convenient laboratory aid for the diagnosis of transmissible spongiform encephalopathy in naturally or accidentally infected animals and humans. (+info)
Recovery of functional response in the nucleus of the solitary tract after peripheral gustatory nerve crush and regeneration.
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Single-unit recording and transganglionic tracing techniques were used to assess the properties of, and inputs to, neurons within the rostral nucleus of the solitary tract (NST) after peripheral gustatory nerve injury and regeneration in adult hamsters (Mesocricetus auratus). Tastant-evoked responses were recorded from 43 neurons in animals in which the ipsilateral chorda tympani (CT) nerve was crushed 8 wk earlier (experimental animals) and from 46 neurons in unlesioned control animals. The 89 neurons were separated into three functional clusters named according to the best stimulus for neurons in the cluster: S, sucrose; N, sodium acetate; and H, HCl or KCl. Stimulus-evoked spike rates across all stimuli were 35.4 +/- 4.4% lower in the experimental hamsters. The largest difference in evoked spike rates occurred for neurons in the H cluster, in which the response to KCl also was delayed relative to normal responses. The response of S-cluster units to sucrose and saccharin was also lower in the experimental animals. The mean response rate and the time course of response of neurons in the N cluster did not differ between the two groups. For each cluster, the spontaneous rates and mean response profiles across eight stimuli were very similar in the experimental and control animals, and the breadth of tuning hardly differed. In both groups, Na+ responses in the N cluster were amiloride sensitive, and responses to the water rinse after stimulation with HCl were common in the S cluster. At 8-20 wk after nerve crush, biotinylated dextran tracing of the CT nerve revealed that the regenerated CT fibers did not sprout outside the normal terminal zone in the NST, but the density of the central terminal fibers was 36.9 +/- 6.35% lower than normal. After CT nerve crush and regeneration, the overall reduction in taste-evoked spike rates in NST neurons is likely a consequence of this change in terminal fibers; this in turn likely results from the known reduction in CT fibers regenerating past the crush site. In the face of this reduction, the normal taste-evoked spike rate in N-cluster neurons requires explanation. The observed recovery of normal specificity could be mediated by a restoration of specific connections by primary afferent fibers peripherally and centrally or by central compensatory mechanisms. (+info)
Limited potential for mosquito transmission of genetically engineered, live-attenuated Venezuelan equine encephalitis virus vaccine candidates.
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In an attempt to improve the current live-attenuated vaccine (TC-83) for Venezuelan equine encephalitis (VEE), specific mutations associated with attenuation of VEE virus in rodent models were identified. These mutations were inserted into full-length cDNA clones of the Trinidad donkey strain of VEE virus by site-directed mutagenesis, and isogenic virus strains with these mutations were recovered after transfection of baby hamster kidney cells with infectious RNA. We evaluated 10 of these strains for their ability to replicate in and be transmitted by Aedes taeniorhynchus, a natural vector of epizootic VEE virus. Two vaccine candidates, one containing a deletion of the PE2 furin cleavage site, the other a combination of three separate point mutations in the E2 glycoprotein, replicated in mosquitoes and were transmitted to hamsters significantly less efficiently than was either parental (wild type) VEE virus or TC-83 virus. Although the attenuated strains were transmitted to hamsters by mosquitoes, after intrathoracic inoculation, there was no evidence of reversion to a virulent phenotype. The mutations that resulted in less efficient replication in, or transmission by, mosquitoes should enhance vaccine safety and reduce the possibility of environmental spread to unintentional hosts. (+info)
Soluble CD40 in the serum of healthy donors, patients with chronic renal failure, haemodialysis and chronic ambulatory peritoneal dialysis (CAPD) patients.
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CD40 and its ligand CD40L are key players in T cell-B cell interaction and T cell-antigen-presenting cell (APC) interaction. Inhibition of CD40-CD40L interaction leads to severe humoral and cellular immunodeficiency. In this study we examined the presence of soluble CD40 (sCD40) in the serum of haemodialysis (HD) patients, CAPD patients, chronic renal failure (CRF) patients and healthy donors in order to evaluate the possible involvement of CD40 in uraemic immunodeficiency. Soluble CD40 was detected in the serum of healthy donors (n = 41) with a mean of 0.14 +/- 0.12 ng/ml and in the urine of healthy donors with a mean of 1.80 +/- 0.74 ng/ml. Soluble CD40 was highly elevated in all patients with impaired renal function. HD patients (n = 22) had up to 100-fold elevated sCD40 levels with a mean concentration of 8.32 +/- 4.11 ng/ml, whereas CAPD patients (n = 10) had considerably lower levels of sCD40 with a mean of 3.58 +/- 2.40 ng/ml. A strong correlation between sCD40 and serum creatinine levels was noted in CRF patients (n = 66). The highly elevated levels of sCD40 may point to the involvement of CD40 and its ligand CD40L in the clinical manifestation of uraemic immunodeficiency. (+info)